Load-dependent systolic parameters

The AB-wk6 group was associated with preserved systolic performance. Accordingly, no difference could be observed in load-dependent systolic parameters (EF, SV, CO) between the AB and the sham group at week 6 (Fig. 12, Table 4). In contrast, in the AB-wk12 and AB-wk18 groups, EF decreased significantly, while SV and CO showed a tendency towards decreased values compared to the corresponding sham groups (Fig. 12, Table 4).

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Figure 12. Representative steady-state pressure-volume (P-V) loops are shown demonstrating in vivo left ventricular (LV) function in sham and aortic banded (AB) rats at different time points. The width of the P-V loops in the AB group at week 6 does not differ from the control’s width. In contrast, at week 12 and 18, the width of the loops becomes substantially smaller in the AB groups, suggesting impaired systolic performance. Furthermore, the P-V loops demonstrate a rightward shift in the AB groups at week 12 and 18, indicating chamber dilatation.

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Table 4. Steady state functional parameters in aortic banded and sham-operated rats at different time points. Values are expressed as mean ± standard error of the mean. AB indicates aortic banding; SBP: systolic arterial blood pressure; DBP: diastolic arterial blood pressure; MAP: mean arterial pressure;

HR: heart rate; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; SV: stroke volume; CO: cardiac output; EF: ejection fraction. **:

P<0.01 vs. age-matched sham. ***: P<0.001 vs. age-matched sham. ##: P<0.01 vs. AB-week 6. ###: P<0.001 AB-week 6. $: P<0.05 vs. AB-week 12.

$$: P<0.01 vs. AB-week 12.

Week 6 Week 12 Week 18

Sham (n=9)

AB (n=13)

Sham (n=9)

AB (n=13)

Sham (n=10)

AB (n=13)

SBP, mmHg 148±4 215±4*** 138±5 215±5*** 150±5 228±4***

DBP, mmHg 116±3 150±2*** 110±4 154±4*** 120±4 170±3***###$$

MAP, mmHg 127±4 172±2*** 119±4 174±4*** 140±4 189±3***##$

HR, beats/min 355±7 369±9 354±5 366±7 379±7 357±5

LVEDV, µl 268±16 305±14 286±23 320±20 283±18 327±14

LVESV, µl 175±15 194±12 178±17 231±11 160±11 241±11***

SV, µl 188±16 173±10 195±11 163±12 175±10 151±15

CO, ml/min 66.7±6.1 62.9±3.0 69.4±4.5 59.4±4.1 66.3±4.4 53.7±5.4

EF, % 58±3 51±2 57±2 44±2** 55±2 41±3***##

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5.1.3.3. Load-independent contractility parameters

In the AB-wk6 group, ESPVR, PRSW and dP/dtmax-EDV increased significantly compared to the sham-wk6 group, indicating increased LV contractility (Fig. 13-16). This contractility augmentation diminished in the AB-wk12 and AB-wk18 groups (Fig. 13-16). Accordingly, the load-independent contractility parameters were not different in the AB-wk12 and AB-wk18 groups compared to sham-wk12 and sham-wk18 groups, but ESPVR and PRSW were significantly decreased compared to the AB-wk6 group (Fig.

16).

Figure 13. Representative pressure-volume loops in the aortic banded and the sham groups at different time points. Original recordings were obtained at different preloads during transient vena cava occlusion. At week 6, the slope of the end-systolic P-V relationship (ESPVR) was steeper in the aortic banded (AB) group, suggesting enhanced LV contractility. In contrast, the slope of the of the end-systolic P-V relationship (ESPVR) did not differ in the aortic banded (AB) group at week 12 and 18 from its corresponding sham group.

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Figure 14. Alterations in preload recruitable stroke work (PRSW) during the progression of pressure overload-induced myocardial hypertrophy. PRSW indicated increased left ventricular (LV) contractility in the aortic banded (AB) group at week 6 compared to the corresponding sham group. The contractility augmentation diminished in the AB groups at week 12 and 18.

53

Figure 15. Alterations in the slope of the maximal systolic pressure increment (dP/dtmax)-end diastolic volume (EDV) relationship during the progression of pressure overload-induced myocardial hypertrophy. dP/dtmax-EDV indicated augmented left ventricular (LV) contractility in the aortic banded (AB) group at week 6 compared to the corresponding sham group. The contractility enhancement diminished in the AB groups at week 12 and 18.

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Figure 16. Left ventricular contractility parameters during the development and progression of pressure overload-induced left ventricular myocardial hypertrophy. Both the slope of the end-systolic pressure-volume relationship (ESPVR) (A), preload recruitable stroke work (PRSW) (B) and the slope of the maximal systolic pressure increment (dP/dtmax)-end diastolic volume (EDV) relationship (C) were increased in the aortic banded (AB) group at week 6 compared to the corresponding sham group, suggesting enhanced left ventricular contractility. This contractility augmentation diminished in the AB groups at week 12 and 18. *: P<0.05. **: P<0.01. ***:

P<0.001.

55 5.1.3.4. Ventricular-arterial coupling

In the AB-wk6 group, the enhanced LV contractility (increased ESPVR) (Fig. 13 and Fig. 16) counterbalanced the elevated afterload (increased Ea) (Table 5), therefore VAC did not differ from the corresponding sham group (Table 5). In contrast, in the AB-wk12 and AB-wk18 groups, the lack of compensatory LV contractility augmentation (reduced ESPVR values compared to AB-wk6) along with the elevated afterload (increased Ea) resulted in contractility-afterload mismatch. Thus, the values of VAC were significantly higher in the AB-wk12 and AB-wk18 groups compared to that of the AB-wk6 group (Table 5).

5.1.3.5. Diastolic parameters

Tau significantly increased in the AB-wk6, AB-wk12 and AB-wk18 groups compared to their corresponding sham groups (Table 5). Furthermore, the slope of EDPVR was also elevated in the AB-wk18 group compared to the sham-wk18 group (Table 5).

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Table 5. Arterial elastance, ventriculo-arterial coupling and indices of diastolic function in aortic banded and sham-operated rats at different time points. Values are expressed as mean ± standard error of the mean. AB indicates aortic banding; Ea: arterial elastance, VAC: ventriculo-arterial coupling Tau: time constant of LV pressure decay according to the Glantz’ method; EDPVR: end-diastolic pressure-volume relationship; *: P<0.05 vs. age-matched sham. **: P<0.01 vs. age-matched sham. ***: P<0.001 vs. age-matched sham. #: P<0.05 vs. week 6. ##: P<0.01 vs. week 6. ###: P<0.001 AB-week 6.

Week 6 Week 12 Week 18

Sham (n=9)

AB (n=13)

Sham (n=9)

AB (n=13)

Sham (n=10)

AB (n=13)

Ea, mmHg/µl 0.75±0.06 1.20±0.08* 0.68±0.05 1.33±0.10*** 0.84±0.05 1.54±0.16***

VAC 0.50±0.08 0.45±0.06 0.54±0.06 0.76±0.08## 0.57±0.10 0.87±0.08###

Tau, ms 14.2±0.4 18.4±0.9** 12.8±0.6 19.4±0. 6*** 13.0±0.3 21.7±1.2***#

EDPVR, mmHg/µl 0.038±0.005 0.038±0.007 0.028±0.004 0.042±0.006 0.014±0.003 0.032±0.004**

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5.2. Investigating the effects of myocardial reverse remodeling from early- versus late-stage left ventricular hypertrophy in male rats

5.2.1. Effect of early and late debanding on echocardiographic parameters In the AB groups, sustained PO led to continuous increment in LV mass, AWTd and PWTd (Fig. 17-18). Both early and late debanding resulted in significant regression of the previously increased LV mass, AWTd and PWTd (Fig. 17-18). To assess the extent of hypertrophy regression in the early- and in the late debanded groups, a ratio was calculated from the parameters measured at the post-debanding and the pre-debanding state. These calculated values were used to compare the effectiveness of reverse remodeling between the early and the late debanded groups. No difference was found in the extent of LV mass (Fig. 19A), AWTd (Fig. 19B) and PWTd (Fig. 19C) regression between the early and late debanded groups.

Figure 17. Representative echocardiographic images during the development of pressure overload-induced left ventricular myocardial hypertrophy and its regression after early and late debanding. Characteristic M-mode echocardiographic recordings at the midpapillary muscle level are shown at week 12 (in case of early debanding) and at week 18 (in case of late debanding) in the sham, the aortic banded (AB) and the debanded groups. Myocardial hypertrophy effectively regressed after early and late debanding as well. AWT: anterior wall thickness, PWT: posterior wall thickness, LVEDD: left ventricular (LV) end-diastolic diameter, LVESD: LV end-systolic diameter, s: systole, d: diastole.

58

Figure 18. Echocardiographic follow-up during myocardial reverse remodeling from early- and late-stage of pathological myocardial hypertrophy. Both in the early and in the late debanded groups, left ventricular (LV) mass (LVmass) (A, D), anterior wall thickness measured in diastole (AWTd) (B, E) and posterior wall thickness measured in diastole (PWTd) (C, F) effectively regressed after pressure unloading. AB indicates aortic banded. *: P <0.05 vs. age-matched sham.

#: P<0.05 vs. age-matched AB.

Figure 19. Direct comparison of left ventricular hypertrophy regression between the early and the late debanded groups. Left ventricular mass (LVmass) (A), anterior wall thickness in diastole (AWTd) (B) and posterior wall thickness in diastole (PWTd) (C) regressed to a similar extent in the early and the late debanding groups.

59

5.2.2. Effect of early and late debanding on pathological hypertrophy markers In the AB groups CD, HW/TL and the expression of the fetal genes (β/α-MHC and ANP) were increased compared to the age-matched control groups (Fig. 20-22).

Both in the early and in the late debanded groups, CD, HW/TL and the expression of the fetal genes (β/α-MHC and ANP) were decreased compared to the age-matched AB groups (Fig. 20A, C, Fig. 21 and Fig. 22A, C). Regarding CD (Fig. 20B), HW/TL (Fig.

20D) and β/α-MHC (Fig. 22B), no differences could be detected in the extent of regression between the early- and the late debanded groups. However, the relative mRNA expression level of ANP decreased to a greater extent in the early debanded compared to the late debanded group (Fig. 22D).

Figure 20. Effect of early and late debanding on macroscopic and microscopic myocardial hypertrophy markers.: Cardiomyocyte diameter (CD) and heart weigth-to-tibial length ratio (HW/TL) were increased in the aortic banded (AB) groups at week 12 and 18 compared to their age-matched sham groups. Both early and late debanding resulted in substantial decrement of CD and HW/TL (A, C). No differences could be observed in the extent of CD and HW/TL regression between the early and the late debanded groups (B, D). *: P<0.05.

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Figure 21. Representative microphotographs demonstrating enlargement of cardiomyocytes in the aortic banded (AB) groups and their regression in both the early and late debanded groups:

Hematoxylin and eosin (magnification 200x, scale bar: 100µm) stained sections are shown. AB indicates aortic banding.

Figure 22. Fetal gene expression during pressure unloading-evoked reverse remodeling. Beta-to-alpha myosin heavy chain ratio (β/α-MHC) and atrial natriuretic peptide (ANP) were increased in the aortic banded (AB) groups at week 12 and 18 compared to the sham groups. Both early and late debanding resulted in substantial decrement of MHC and HW/TL (A, C). Although, β/α-MHC showed similar extent of regression in the debanded groups (B), ANP normalized to a greater extent in the early debanding compared to the late debanded group (D). *: P<0.05.

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5.2.3. Effect of early and late debanding on reactive myocardial fibrosis

In the early debanded group, interstitial and perivascular fibrosis decreased compared to the age-matched AB group (Fig. 23A, C and Fig. 24). However, in the late debanded group, interstitial and perivascular fibrosis remained increased, and it did not differ from the age-matched AB group (Fig. 23A, C and Fig. 24). Accordingly, the extent of both interstitial and perivascular fibrosis showed significantly higher levels in the late debanded group compared to the early debanded group (Fig.23B, D).

Figure 23. Regression of interstitial and perivascular fibrosis after pressure unloading at different time points. Increased interstitial and perivascular fibrosis were observed in the aortic banded (AB) groups at week 12 and 18. Regression of reactive interstitial and perivascular fibrosis could be detected only in the early debanded group, while the collagen accumulation persisted in the late debanding group (A, C). Accordingly, robust differences were observed in interstitial and perivascular fibrosis between the two debanded groups (B, D). *: P<0.05.

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Figure 24. Representative histological photomicrographs of interstitial and perivascular fibrosis demonstrating regression of reactive fibrosis in the early debanded group and persisting collagen accumulation in the late debanded group. Representative photomicrographs of picrosirius red staining are shown demonstrating interstitial fibrosis (A, C) (magnification 50x, scale bar: 200µm) and perivascular fibrosis (B, D) (magnification 100x, scale bar: 100µm). AB:

aortic banding.

In the AB groups increased mRNA levels of CTGF were detected (Fig. 25A). The protein level of CTGF showed a strong tendency towards increased values in the AB-wk12 group compared to the age-matched sham group. Furthermore, in the AB-wk18 groups, the protein level of CTGF was significantly increased compared to the corresponding sham group (Fig. 25C). The enhanced expressional (mRNA and protein)

63

level of CTGF was normalized in both the early and the late debanding groups (Fig. 25A, C). No differences could be observed in the extent of CTGF regression between the two debanded groups (Fig. 25B, D).

Figure 25. Effect of early and late debanding on connective tissue growth factor (CTGF) mRNA and protein expression. CTGF mRNA expression was increased in the aortic banded groups at week 12 and 18 (A). Furthermore, protein expression of CTGF was also significantly higher in the AB-wk18 group compared to its corresponding sham group (C). Both early and late debanding effectively normalized the AB-associated alterations in CTGF mRNA and protein expression (A, C). No differences could be observed in the extent of CTGF normalization in the early and late debanding groups (B, D) GAPDH: glyceraldehyde 3-phosphate dehydrogenase. *:

P<0.05.

5.2.4. Effect of early and late debanding on hemodynamic parameters 5.2.4.1. Arterial loading and meridional wall stress

In the AB groups SBP, DBP, MAP and σ were increased compared to the age-matched sham groups (Table 6). The extent of SBP, DBP and MAP increment was equal between

64

the AB groups at different time points (Table 7). However, σ increased to a significantly greater degree in the AB-wk12 and AB-wk18 groups compared to the AB-wk6 group (Table 7). In the early and late debanded groups, SBP, DBP, MAP and σ were decreased compared to the age-matched AB groups (Table 6). The extent of afterload and wall stress reduction did not differ between the two debanded groups (Table 7).

5.2.4.2. Load-dependent systolic parameters

In the AB-wk12 and AB-wk18 groups, EF was significantly reduced, while SV and CO showed a tendency towards decreased values when compared to their age-matched control groups (Table 6, Fig. 26A and Fig. 27).Both in the early and in the late debanded groups, EF, SV and CO were increased compared to the age-matched AB groups (Table 6, Fig. 26A and Fig. 27). No differences were found regarding the extent of EF, SV and CO improvement between the early and late debanded groups (Table 7, Fig.26B and Fig.

27).

Figure 26. Changes in ejection fraction (EF) during the development of pathological myocardial hypertrophy and its regression by early and late debanding. Both the early debanding and the late debanding groups were associated with increased EF (A). The extent of EF improvement was similar between the two debanded groups AB indicates aortic banding. *:

P<0.05.

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Figure 27. Representative left ventricular (LV) pressure-volume (P-V) steady-state loops demonstrating the effect of early and late debanding on left ventricular function. In the aortic banded (AB) groups at week 12 and 18, the width of the loops was substantially smaller compared to the corresponding age-matched sham groups, suggesting impaired LV systolic function. Both early and late debanding effectively increased the width of the P-V loops, indicating that pressure unloading therapy performed either at early or late time points successfully improved LV systolic performance.

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Table 6. Hemodynamic parameters in the aortic banded (AB) groups at different time points and in the early and the late debanded groups. Values are expressed as mean ± standard error of the mean. AB indicates aortic banding; SBP: systolic arterial blood pressure; DBP: diastolic arterial blood pressure; MAP: mean arterial pressure; σ: meridional wall stress, HR: heart rate; dP/dtmax: maximal slope of systolic pressure increment; dP/dtmin: maximal slope of diastolic pressure decrement; LVEDP: left ventricular (LV) end-diastolic pressure; LVESP: LV end-systolic pressure; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; SV: stroke volume; CO: cardiac output. *: P<0.05 vs. age-matched Sham, #: P<0.05 vs. age-matched AB.

12 week 18 week

HR, beats/min 356±6 365±7 357±7 381±7.5 359±4.7* 351±5.1*

dP/dtmax, mmHg/s 8291±353 10579±436* 9467±483 9670±359 11062±505 9522±267#

dP/dtmin, mmHg/s -9220±313 -9633±277 -9887±563 -10266±328 -8838±437* -8907±237*

LVEDP, mmHg 4.0±0.4 7.1±0.9* 5.3±0.7 5.4±0.4 7.8±1.0* 5.8±0.4

LVESP, mmHg 133±3 202±4* 161±4*# 147±5 209±5* 168±5*#

LVEDV, µl 340±19 357±18 340±16 329±19 372±19 362±13

LVESV, µl 148±13 201±11* 166±10 150±10 216±12* 166±13#

SV, µl 192±10 156±12 174±10 178±11 156±17 196±14

CO, ml/min 68.6±4.1 56.7±4.3 61.4±3.0 68.3±4.4 55.9±5.8 68.8±5.0

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Table 7. Comparison of pressure overload-induced alterations in the aortic banded groups at different time points and during myocardial reverse remodeling after early and late debanding. The values of the aortic banded (AB) and the debanded groups were normalized to the corresponding sham groups. These calculated values are expressed as mean ± standard error of the mean. SBP indicates systolic arterial blood pressure; DBP: diastolic arterial blood pressure; MAP: mean arterial pressure; Ea: arterial elastance, σ: meridional wall stress, LVEDP: left ventricular end-diastolic pressure; LVESP:

LV end-systolic pressure; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; SV: stroke volume; CO: cardiac output. *: P<0.05 vs.

age-matched Sham.

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5.2.5.3. Load-independent contractility parameters

In the AB-wk12 and AB-wk18 groups, neither ESPVR nor PRSW differed from their corresponding control groups (Fig. 28, 29A, 30A). Similarly, no alteration could be observed in ESPVR and PRSW after early or late debanding. (Fig. 28, 29A, 30A).

Figure 28. Representative pressure volume loops demonstrating characteristic alterations in the aortic banded (AB) groups and in the early and late debanded groups. Original recordings were obtained at different preloads during transient vena cava occlusion. The slope of end-systolic pressure volume relationship (ESPVR) did not differ among the study groups. In contrast, the slope of the end-diastolic pressure volume relationship (EDPVR) was increased in the AB groups, suggesting impaired myocardial stiffness. Increased EDPVR was normalized in the early debanded group, while it persisted in the late debanded group.

69

Figure 29. Alterations in preload recruitable stroke work (PRSW) after early and late debanding. PRSW did not differ in the aortic banded (AB) groups at week 12 and week 18 from the corresponding control groups (A). Similarly, no alterations could be detected in PRSW neither in the early nor in the late debanding groups (A, B). *: P<0.05.

5.2.5.4. Ventriculo-arterial coupling

In the AB-wk12 and AB-wk18 groups, ESPVR did not differ from the control groups (Fig. 30A). Therefore, at these time points, increased Ea (Fig. 30C) was not compensated by enhanced contractility, which led to impaired VAC (Fig. 30E). Both in the early- and in the late debanded groups, reduction of Ea efficiently normalized the relation of the LV contractility and the connecting arterial system and resulted in adequate VAC values (Fig.

30E). No differences could be observed in the extent of VAC normalization between the early and the late debanded groups (Fig. 30F).

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Figure 30. Ventriculo-arterial coupling (VAC) during the development of aortic banding (AB)-induced pathological myocardial hypertrophy and during its regression by early and late debanding. The slope of end-systolic pressure-volume relationship (ESPVR) did not differ among the study groups (A-B). In contrast arterial elastance (Ea) was significantly increased in the aortic banded (AB) groups at week 12 and 18 (C). Both early and late debanding effectively reduced the increased Ea (C). The extent of Ea normalization was similar between the two debanded groups (D). The lack of compensatory contractility augmentation led to impaired ventriculo-arterial coupling (VAC) in the AB groups at week 12 and 18. By decreasing Ea, both early and late debanding resulted in the normalization of VAC. No differences could be detected in the improvement of VAC between the early and the late debanded groups. *: P<0.05.

5.2.5.5. Diastolic parameters

Tau was prolonged in all the AB groups (Fig. 31A). Furthermore, the slope of EDPVR was also increased in AB groups at week 12 and 18 compared to sham (Fig. 31C). In the early debanded group, the prolonged active relaxation was effectively shortened, and Tau

71

did not differ from the control’s level (Fig. 31A). In the late debanded group, Tau was also decreased compared to the age-matched AB group. However, the extent of improvement was smaller compared to the early debanded group and Tau remained significantly prolonged in the late debanded group when compared the control’s level (Fig. 31A-B). Furthermore, in the early debanded group, LV stiffness was also decreased compared to the corresponding AB group (Fig. 31C). In contrast, no significant differences could be observed in EDPVR between the late debanded group and the AB-wk18 group (Fig. 31C). Therefore, the extent of improvement in LV stiffness was also significantly smaller in the late debanded group than that observed in the early debanded group (Fig. 31D).

Figure 31. Diastolic function during the development of aortic banding (AB)-induced LVH and its regression by early and late debanding. Prolongation of the active relaxation time constant (Tau) and increased myocardial stiffness (assessed by the elevated slope of the end-diastolic pressure-volume relationship [EDPVR]) were detected in the AB groups at week 12 and 18 (A, C). Debanding the aortic constriction at an early time point effectively improved both aspects of diastolic function (A, C). In contrast, late debanding failed to normalize diastolic dysfunction. Accordingly, robust differences were observed between the early and the late debanding groups (B, D). *: P<0.05.

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5.3. Investigation of sex differences during the development of pressure overload-induced left ventricular hypertrophy

5.3.1. Effect of sex on the temporal development of left ventricular hypertrophy LV mass index was increased in both male and female AB rats from week 3 until the end of the experimental period (Fig. 32 and Fig. 33A). RWT revealed concentric LV geometry at week 6 in both male and female AB groups (Fig 32 and Fig. 33C).

Concentric geometry was also observed in the female AB rats at week 12. However, at week 12, RWT substantially decreased in male rats, leading to eccentric LV geometry

Concentric geometry was also observed in the female AB rats at week 12. However, at week 12, RWT substantially decreased in male rats, leading to eccentric LV geometry

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